Diagnostic devices and methods of use

ABSTRACT

The present invention relates to methods of diagnosing samples as well as various microfluidic, microcentrifuge and microfilter devices. In one embodiment, the present invention provides a method of diagnosing neurodegenerative diseases using mitochondrial and/or platelet samples. In another embodiment, the present invention provides a microfluidic device that selectively captures and analyzes a desired amount of target biological particle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority as a continuation ofU.S. patent application Ser. No. 15/254,146, filed Sep. 1, 2016, whichis a continuation of U.S. Pat. No. 9,463,458, filed Aug. 25, 2011, whichis the National Phase of International Application No.PCT/US2010/025964, filed Mar. 2, 2010, which designated the UnitedStates and that International Application was published under PCTArticle 21(2) in English, which also includes a claim of priority toU.S. Provisional Patent Application Ser. No. 61/156,717, filed Mar. 2,2009, and 61/156,734, filed Mar. 2, 2009, the entirety of which ishereby incorporated by reference for all purposes.

FIELD OF THE INVENTION

The invention relates to methods of performing diagnostic analyses ontissue samples as well as microfluidic and microfilter devices.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Originally developed for producing large-scale integrated circuits,microfabrication technology has also spurred research and developmentefforts to manipulate and analyze complex biological fluids at the nano-and microliter scale. The development of miniaturized diagnostic devicesfor the clinical analysis of blood and other body fluids is an importantapplication of this emerging technology.¹⁻⁶ For example, point-of-useblood glucose monitoring systems have demonstrated the ability ofminiaturized clinical chemistry technology to improve patient care andhealth care delivery methods.^(7,8)

Many current diagnostic technologies require sample preparation prior toanalysis, and microfluidic devices can be engineered with an integratedisolation process upstream of the analyte detection region. For example,glucose test strips isolate plasma from whole blood with glass fiberfilters or microporous membranes.⁹ Though researchers have reported manylab-on-a-chip diagnostic advances, few results relevant to themicrofluidic fractionation of samples are reported.¹⁰ For example, Brodyet al.¹¹ suggested separating plasma from whole blood using amicrofabricated filter device, and reported the filtration of asuspension of microspheres. Wilding et al.¹² demonstrated usingmicrofilters to measure white blood cells for genetic analysis, andDuffy et al.¹³ proposed centrifugation on a rotating “lab disc.” Still,the current state of the art lacks a device capable of isolating foroptical visualization a desired number or numerical range of biologicalparticles from a liquid biological sample. Such a device would be oftremendous utility in the point-of-care diagnostics environment byenabling rapid, low-cost diagnoses.

One possible application for microfluidic devices is their use inconjunction with new and reliable biomarkers for diagnosis and treatmentof conditions and diseases. For example, recent research has indicatedthat certain functional declines specific to neurological conditionssuch as Alzheimer's Disease can be detected in peripheral cells,including platelets. Recent results have also indicated that these samechanges can also be detected in platelets from subjects who have beendiagnosed with mild cognitive impairment (MCI), often a preclinicalprecursor to full-blown AD, marked most often by mild memory loss(“monosymptomatic progressive amnesia”).

Microfilters are well suited for microfluidic sample preparation, asthey are compatible with current microfabrication technologies, andfiltration can be accomplished via pressurization, capillary action, orother induced flows. Further, the precise dimensional and geometriccontrol afforded by micromachining enables the development of optimumfilter designs that are not possible with traditional membranefiltration. For example, hollow fiber membranes (commonly used forplasma separation) have bulk flow channel diameters of 200 to 400 μm,while microfluidic channels are readily constructed at dimensionscommensurate with blood cells (red blood cells typically measure from 6to 7 μm in diameter, platelets 1-2 μm). Also, microfilter devices can befabricated with precise pore dimensions and geometry; in contrast, poresize and geometry in microporous membranes are often heterogeneous.Finally, microfilter devices fabricated with optically transparentmaterial can enable direct visualization of target pathology or labelingof such.

SUMMARY OF THE INVENTION

Various embodiments include a microfluidic device, comprising an upperplate, comprising an injector port in fluid communication with anoptical measurement chamber; engaged with a lower plate, comprising oneor more filter channels, where the surface of the optical measurementchamber abutting the lower plate is in fluid communication with the oneor more filter channels. In another embodiment, the upper platecomprises more than one injector port. In another embodiment, the upperplate comprises more than one optical measurement chamber. In anotherembodiment, the one or more filter channels are arranged in rows ofparallel lines which in any two adjacent rows slope in oppositedirections. In another embodiment, the one or more filter channels areless than 1.0 μm wide. In another embodiment, the lower plate is engagedwith a capillary eluent chamber that enhances filter flow throughcapillary force. In another embodiment, a flow channel attaches theinjector port to the optical measurement chamber. In another embodiment,the lower plate is made of glass. In another embodiment, the upper plateis made of glass. In another embodiment, one or more temperaturecontrollers adapted to regulate temperature within the opticalmeasurement chamber. In another embodiment, the optical measurementchamber is configured to retain molecules on the basis of affinity,size, charge or mobility. In another embodiment, the optical measurementchamber is configured to retain within 5% of a target number ofbiological particles. In another embodiment, the optical measurementchamber is configured to retain within 2% of a target number ofbiological particles. In another embodiment, the optical measurementchamber is configured to retain within 1% of a target number ofbiological particles. In another embodiment, the device is adapted suchthat the flow of fluid from the injector port to the optical measurementchamber is regulated by pressure, valves, or other similar components.

Other embodiments include a microfluidic kit for the diagnosis ofAlzheimer's Disease and/or mild cognitive impairment, comprising amicrofluidic diagnostic device; and instructions for use.

Other embodiments include a kit for the diagnosis of a neurodegenerativedisease, comprising a centrifugal diagnostic device comprising anoptical microcentrifuge tube insert and/or cuvette; and instructions foruse. In another embodiment, the neurodegenerative disease is Alzheimer'sDisease and/or mild cognitive impairment.

Various embodiments include a method of diagnosing a neurodegenerativedisease, comprising obtaining a sample from a subject; quantifying theamount or activity of a target moiety in the sample by transferring thesample to the device described in claim 1 herein, and comparing theamount or activity of the target moiety in the sample with the amount oractivity of the target moiety in a standard, wherein a change in amountor activity of the target moiety relative to the standard indicates thatthe subject has or will develop a neurodegenerative disease. In anotherembodiment, the sample comprises an unprocessed sample. In anotherembodiment, the sample comprises a peripheral tissue sample. In anotherembodiment, the target moiety comprises mitochondrial protein. Inanother embodiment, the sample does not contain mitochondrial isolateand/or selection. In another embodiment, the sample has not been exposedto a high-energy disruption technique. In another embodiment, the samplehas not been exposed to sonication, nitrogen cavitation and/or lysisprocedure. In another embodiment, quantifying the amount or activity ofa target moiety comprises determining the cytochrome oxidase activity inthe tissue sample. In another embodiment, quantifying the amount oractivity of a target moiety comprises determining the amount of amyloidprecursor protein present in the unprocessed sample. In anotherembodiment, the sample comprises plasma. In another embodiment, thesample comprises platelet-rich plasma (PRP). In another embodiment, thesubject is mammalian. In another embodiment, the subject is a human. Inanother embodiment, the neurodegenerative disease is at least one ofAlcoholism, Alexander's disease, Alper's disease, Alzheimer's Disease,amyotrophic lateral sclerosis (ALS), Ataxia telangiectasia, Battendisease, Bovine spongiform encephalopathy (BSE), Canavan disease,Cerebral palsy, Cockayne syndrome, Corticobasal degeneration,Creutzfeldt-Jakob disease, Frontotemporal lobar degeneration,huntington's disease, HIV-associated dementia, Kennedy's disease,Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Josephdisease, Multiple System Atrophy, Multiple sclerosis, Narcolepsy,Niemann Pick disease, PD, Pelizaeus-Merzbacher Disease, Pick's disease,Primary lateral sclerosis, Prion diseases, Progressive SupranuclearPalsy, Refsum's disease, Sandhoff disease, Schilder's disease, Subacutecombined degeneration of spinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease, Spinocerebellar ataxia, Spinalmuscular atrophy, Steele-Richardson-Olszewski disease, and Tabesdorsalis. In another embodiment, the targeting moiety comprisesmitochondrial translocase subunit. In another embodiment, the targetingmoiety comprises amyloid precursor protein, or fragments thereof,complexed with mitochondrial protein.

Other embodiments include a method of diagnosing a neurodegenerativedisease, comprising obtaining a sample from a subject; quantifying theamount or activity of a target moiety in the sample by transferring thesample to a centrifugal device; and comparing the amount or activity ofthe target moiety in the sample with the amount or activity of thetarget moiety in a standard, wherein a change in amount or activity ofthe target moiety relative to the standard indicates that the subjecthas or will develop a neurodegenerative disease. In another embodiment,the centrifugal device comprises an optical microcentrifuge tube insertand/or cuvette.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with an embodiment herein, a schematic ofa microfluidics device that will allow densitometric measurement of thestained suspended-cell sample. The general design may bemicrofabrication technology, containing sandwich design where surfaceetched features become internal features when assembled. This device maycontain two etched glass plates that, when assembled and bonded, form aninternal network of interconnected channels and chambers. (A) disclosesthe upper plate in both a top view 100 and side views 101. The upperplate contains an injector port 102 for injection of the samplesuspended in buffer, a flow channel 103 leading to a optical measurementchamber 104 that lies over filter channels 107 etched into the lowerplate. (B) discloses the lower plate in both a top view 105 and sideviews 106. In one embodiment, six to eight of these devices may bearrayed on a chip designed to the format of 96-well plates, to be readin a standard microplate reader.

FIG. 2 depicts, in accordance with an embodiment herein, a schematic ofa microfluidics device that will allow densitometric measurement ofstained suspended-cell sample. The general design may bemicrofabrication technology, containing sandwich design where surfaceetched features become internal features when assembled. (A) disclosesthe upper plate in both a top view 100 and side views 101. The upperplate contains an injector port 102, drilled through, (e.g., for 21G=0.8 mm outer diameter needle). The upper plate also contains a flowchannel 103, 1 mm wide×500 μm high, leading to an optical measurementchamber 104 overlaying filter channels 107 in traverse flow, with lengthvariable. (B) discloses the lower filter plate in both a top view 105and side views 106, with the lower filter plate dimensionally smallerthan the upper plate. The filter channels 107 in the lower filter plateare in a herringbone pattern of shallow transverse flow filter channels,less than 1.0 μm wide, with depth indeterminate. In one embodiment,there may also be a lower capping plate, featureless and dimensionallyidentical to the upper plate, including a capillary eluent chamber 108formed by the lower capping plate or wicking absorbent.

FIG. 3 depicts, in accordance with an embodiment herein, an insertdesign to allow densitometric measurement of stained suspended cellsample using a microcentrifuge. The insert may act similar to amicrocentrifuge tube insert where the stained particles, such asplatelets, may reliably pellet into the optical pocket of the insert forstandardized optical measurement. The figure depicts a tube 110 withtypical dimensions of a 1.5 mL tube, an optical measurement chamber 104for the insert, which may have, for example, inner dimensions of 2 mmwidth by 14 mm, 0.5-1.0 mm diameter. The figure also depicts a supportfin 109 which may be, for example, about 0.85 mm thick. In oneembodiment described in further detail herein, the staining intensity ofthe platelets will decrease in Alzheimer's disease patients, and inpre-symptomatic subjects, and one will be able to reliably measure thisdecrease.

FIG. 4 depicts, in accordance with an embodiment herein, an insertcuvette 111 design as an add-on to permit an insert read-out in astandard spectrophotometer. The cuvette, in one embodiment, may includea cuvette optical window 112. In accordance with another embodiment, thedesign may also include a mounting slot 113 to capture and orient thesupport fin 109 of the insert. In another embodiment, the add-on canenable read-out in a standard microplate reader.

FIG. 5 depicts, in accordance with an embodiment herein, a flow chart ofthe overall method of quantification of a target moiety from a sample.In one embodiment, the sample may be obtained from the subject 200,followed by staining of the sample 201, then transfer to a microfluidicdevice 202, where the carrier solution may pass through while stainedsample are retained 203, and then finally measuring the optical densityand comparing the density to a standard 204. In another embodiment, thesample may be obtained from the subject 200, followed by staining of thesample 201, then transfer to a centrifugal device 205, centrifuged toinduce sedimentation of stained sample 206, and then finally measuringthe optical density and comparing the density to a standard 207.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used herein, the term “diagnose” or “diagnosis” refers to determiningthe nature or the identity of a condition or disease. A diagnosis may beaccompanied by a determination as to the severity of the disease.

As used herein, “prognostic” or “prognosis” refers to predicting theoutcome of a disease.

As used herein, “AD” means Alzheimer's Disease.

As used herein, “MCI” means mild cognitive impairment.

As used herein, the term “PD” means Parkinson's Disease.

As used herein, “fluid connection” means a connection comprising aliquid connector, such as, for example, water, blood, plasma, and thelike.

As used herein, an “unprocessed sample” refers to a sample that has notbeen exposed to high-energy disruption techniques such as sonication ornitrogen cavitation. As an example, although the term is in no way solimited, an unprocessed sample containing platelet rich plasma may notbe exposed to the filtration, purification and/or isolation of themitochondria itself before being analyzed for mitochondrial enzymedysfunction.

As used herein, the term “herringbone” pattern refers to a pattern madeup of rows of parallel lines which in any two adjacent rows slope inopposite directions.

As used herein, upper and lower plates of a microfluidic device may alsorefer to different sides or areas of a single attached device. Forexample, although in way limiting, a microfluidic device with an upperand lower plate may refer to a single attached device with one sidecontaining an injector port and the opposite side containing variouschannels.

As used herein, “microfluidics” refers to a device that allows for flowof fluid, suspensions or suspended particles at various dimensions andscales of size.

Microfluidics and Flow Devices

As disclosed herein, in accordance with various embodiments herein, theinventors created microfluidic devices with filter channels 107 placedso as to be fluidly connected to an optical measurement chamber 104 whenthe components are joined together. The filter channels 107 can allowliquid to flow from the optical measurement chamber 104 while blockingthe flow of, for example, suspended blood cells. In another embodiment,the microfluidic device is described in FIGS. 1, 2, 3 and/or 4 herein.

Embodiments of the invention include a microfluidic diagnostic device.In certain embodiments, the device can isolate biological particles fromliquid samples. In certain embodiments the particles can be, forexample, erythrocytes, leukocytes, platelets, fibroblasts, culturedcells, and the like. Likewise, in certain embodiments, the liquid samplecan be whole blood, whole blood fractions, plasma, urine, lymph, broth,media, or the like.

Embodiments of the invention can be fabricated from any suitablematerial, such as, for example, plastic, glass, silica, composites, orthe like.

In embodiments that include plastic materials, the plastic materials caninclude, for example, thermoplastics, such as, for example,acrylonitrile butadiene styrene plastics (ABS), acetals, acrylic(Perspex), acrylo-nitrile (nylon), cellulosics, fluoroplastics,high-density polyethylene (HDPE), low-density polyethylene (LDPE),Noryl, polyarylates, polyarylsulfones, polybutylenes, polybutyleneterepthalate (PBT), polycarbonates, polyesters, polyetherimides,polyetherketones, polyethylene (polythene), polypropylene, polyallomers,polyethylene terephalate, polyimides, polyamide-imides, poly vinylacetate (PVA), poly vinyl chloride (PVC), polystyrene, polysulfones,Styrene, ABS PTFE (Teflon), and the like.

In embodiments that include plastic materials, the plastic materials canbe, for example, thermosets, such as, for example, alkyd polyesters,allyls, bakelite, epoxy, melamine, phenolics, polybutadienes, polyester,polyurethane, silicones, ureas, and the like. Likewise, the plasticmaterials can include bioplastics. Bioplastics are a form of plasticsderived from renewable biomass sources, such as vegetable oil, cornstarch, pea starch, or microbiota, rather than traditional plastics thatare often derived from petroleum. Types of bioplastics suitable for usewith embodiments of the invention include, for example, polylactide acid(PLA) plastics, poly-3-hydroxybutyrate (PHB), polyamide 11 (PA 11),bio-derived polyethylene, and the like.

In certain embodiments, the device can be composed of, for example, asingle component. In certain embodiments, the device can be composed ofmultiple components, such as, for example, an upper plate and lowerplate. In some embodiments, the multiple components can be made from thesame or different materials. In certain embodiments, the components canbe homogenous or heterogenous, and can include, for example, channels,ducts, chambers, voids, ports, and the like. In heterogenous embodimentsthe channels, ducts, chambers, voids, ports, and the like, can be ofconsistent size, such as, for example, 1 μm in depth or diameter, or 2μm in depth or diameter, or 4 μm in depth or diameter, or 10 μm in depthor diameter, or more, or the like. Likewise, in heterogenous embodimentsthe channels, ducts, chambers, voids, ports, and the like, can range insize, such as, for example, between 1 and 3 μm in depth or diameter,between 3 and 6 μm in depth or diameter, between 6 and 10 μm in depth ordiameter, or more, or between 1 and 2 mm in depth or diameter, orbetween 2 and 5 mm in depth or diameter, or between 5 and 10 mm in depthor diameter, or more, or the like.

In some embodiments composed of multiple components, the multiplecomponents can be sealed to form a single unit. In certain embodiments,a chamber of the device can capture a predetermined number, amount, orvolume of the target biological particle. Likewise, certain embodimentsof the device can maintain the capture of a predetermined number oramount of the target biological particle. Likewise, in certainembodiments a chamber of the device can capture within 0.1%, 0.5%, 1%,2%, 4%, 7%, 10%, 15%, or 20% or any other desirable amount of apredetermined number or amount of particles.

In certain embodiments, the predetermined number or amount can bewithin, for example, a range, such as from 0 to 100 particles, or from100 to 1000 particles, or from 1000 to 10,000 particles, or from 10,000to 100,000 particles, or from 100,000 to 1,000,000 particles, or from1,000,000 to 5,000,000 particles, or within any of these ranges, ormore, or the like.

In some embodiments the optical measurement chamber 104 can be opticallytransparent, such that the biological particles within the chamber canbe visualized. In certain embodiments the visualization can be achievedvia any suitable means, such as, for example,densitometric/spectrophotometric readers, luminescent/fluorometricreaders, or the like. In certain embodiments, the size and configurationof the measuring chamber can be optimized for a particular targetbiological particle.

Embodiments of the invention can include at least one filter channel 107arranged to drain liquid from the optical measurement chamber 104.

In an embodiment, the present invention provides a device comprising:

(a) an upper plate, including an injector port 102 in fluid connectionwith an optical measurement chamber 104; and;

(b) a lower plate, including at least one filter channel 107;

(c) wherein the surface of the optical measurement chamber 104 abuttingthe lower plate is in fluid connection with the at least one filterchannel 107.

In an embodiment, the present invention provides a method of diagnosing,for example, the presence, absence, or severity of a disease. Likewise,in some embodiments the invention provides a method of diagnosing thelikelihood of developing or not developing a disease. In certainembodiments, the disease can be, for example, AD, PD, otherneurodegenerative diseases, or the like. In certain embodiments, themethod can include providing a biological sample, manipulating thesample so as to be able to visualize certain characteristics of thesample, loading the sample into a device of the present invention, andvisualizing the sample.

Advantages of the design include utilization of easily-attainable,low-volume blood sample that can be processed and measured in <2 hours.Also, the novel microfluidics measurement device enables rapiddensitometric (or fluorescent or luminescent) measurement of labeledsample. The reaction product in device is measurable in a microplatereader after simple reaction steps—designed for possible use in basicclinical labs, with basic equipment, by minimally trained lab personnel.Various embodiments may measure a cellular function that has beenrepeatedly found deficient in Alzheimer's Disease brain, for example,platelets and other tissues, and measure it with increased sensitivityin whole, unlysed cells—in other words, unprocessed samples, not relyingon mitochondrial isolation, which is the largest confound inconventional ETC enzyme studies. The device, in particular, has broadapplication in other diseases, as it is usable for a broad range ofdyes, reactions, and probes, including antibody-based techniques, andcan be modified for different cell types (e.g., lymphocytes, culturedlines) in clinical and research applications.

Diagnostic Methods Using Mitochondria

As disclosed herein, recent research has indicated that themitochondrial electron transport chain (ETC) enzymes are functionallydeficient in AD as well as mild cognitive impairment (MCI). Thesespecific deficiencies (particularly in the enzyme cytochrome oxidase)have been found in brain and in platelets from AD patients, as well asin other peripheral tissues (that are somewhat less accessible; e.g.,muscle). The inventors have found a significant decrease in the functionof cytochrome oxidase (C.O.) in platelet mitochondria isolated fromsubjects with MCI, supporting the view that interference withmitochondrial function is an early and detectable systemic event in ADpathophysiology which can be leveraged as a biomarker of the disease.

In an embodiment, the present invention provides a method of diagnosinga neurodegenerative disease, comprising:

(a) providing a tissue sample from a subject;

(b) quantifying the amount or activity of a target moiety in the cellsof said sample; and

(c) comparing the amount or activity of the target moiety in the cellsof the sample with the amount or activity of the target moiety in astandard, wherein a change in the amount or activity of the targetmoiety in the cells of the sample relative to the standard indicatesthat the subject has a neurodegenerative disease.

In certain embodiments, the neurodegenerative disease can be, forexample, Alcoholism, Alexander's disease, Alper's disease, AD, ALS,Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,Frontotemporal lobar degeneration, HD, HIV-associated dementia,Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, PD, Pelizaeus-Merzbacher Disease, Pick's disease, Primarylateral sclerosis, Prion diseases, Progressive Supranuclear Palsy,Refsum's disease, Sandhoff disease, Schilder's disease, Subacutecombined degeneration of spinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),Spinocerebellar ataxia (multiple types with varying characteristics),Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabesdorsalis.

In some embodiments, the tissue sample can comprise, for example,amniotic fluid, blood, cerebro-spinal fluid, plasma, serum, synovialfluid, other biofluids, or the like. Certain embodiments can utilizefractionated samples, such as, for example, blood fractions, plasmafractions, or the like. Some embodiments can utilize platelet-richplasma (PRP). In embodiments utilizing fractionated samples, thefractionation can be accomplished using, for example, differentialcentrifugation, or the like. In certain embodiments, the tissue samplecan be obtained from the subject via, for example, amniocentesis, ablood draw, a spinal tap, or the like.

In some embodiments, the tissue sample can comprise, for example, musclecells, muscle fibers, or muscle tissue, such as cardiac muscle tissue,smooth muscle tissue, or skeletal muscle tissue, or the like. In certainembodiments, the tissue sample can be obtained via, for example, abiopsy, or the like.

In certain embodiments, the subject can be a mammal, such as, forexample, a marsupial, a monotreme, a placental mammal, or the like. Inembodiments utilizing a placental mammal, the placental mammal can be,for example, a human, or the like. In some embodiments, quantifying theamount or activity of a target moiety can be accomplished via, forexample, histochemical techniques, immunochemical techniques, acombination thereof, or the like.

In certain embodiments, quantifying the amount or activity of a targetmoiety can mean, for example, quantifying the presence of a protein,such as, for example, amyloid beta (Aβ), amyloid precursor protein(APP), mitochondrial translocase (subunit TOMM40), the APP-TOMM40complex, or the like. In some embodiments, quantification can beachieved with the use of antibodies reactive to the target moiety.

In certain embodiments, quantifying the amount or activity of a targetmoiety can mean, for example, assessing enzyme activity. In someembodiments, this can involve, for example,densitometric/spectrophotometric assessment, fluorometric assessment,histochemical labeling, or the like.

In certain embodiments, the standard amount or activity of a targetmoiety can be determined from, for example, patient samples, literature,computer databases, or a combination thereof, or the like. In someembodiments, the change in amount or activity of a target moiety can be,for example, an increase or a decrease.

In certain embodiments, the cells in which the amount or activity of atarget moiety is to be tested can be, for example, anymitochondria-containing cell, such as thrombocytes, or the like.

Certain embodiments of the invention utilize diagnostic test formatssuch as, for example, cuvettes, multi-well plates, microfilter devices,or the like. In some embodiments the multi-well plates can include, forexample, 4 wells, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 192wells, 384 wells, 768 wells, 1536 wells, or more, or the like. In someembodiments the microfilter device can include, for example, a fillport, an optically transparent measurement chamber, and at least onedrainage channel. In some embodiments the microfilter device can befabricated from plastic, glass, silica, or the like.

Kits

Embodiments of the invention are also directed to a kit, both fordiagnosing the presence of, absence of, severity of, or likelihood ofdeveloping or not developing a neurodegenerative disease, as well aspreparation and use of a microfluidic and centrifugal device. The kit isan assemblage of materials or components, suitable for performing amethod of the invention. Thus, in some embodiments the kit describes amethod of the invention, as described above or in the followingexamples.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of diagnosing neurodegenerative diseases. Inone embodiment, the kit is configured particularly for the purpose ofdiagnosing AD. In another embodiment, the kit is configured particularlyfor the purpose of diagnosing PD. Or, in some embodiments, the kitcontains a device including upper plate and lower plate with an injectorport 102 in fluid communication with an optical measurement chamber 104,as described above.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to diagnose the presence of AD in a human. Optionally, the kitalso contains other useful components, such as, diluents, buffers,pharmaceutically acceptable carriers, syringes, catheters, applicators,pipetting or measuring tools, bandaging materials or other usefulparaphernalia as will be readily recognized by those of skill in theart.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive devices and thelike. The packaging material is constructed by well known methods,preferably to provide a sterile, contaminant-free environment. Thepackaging materials employed in the kit are those customarily utilizedin diagnostic assays. As used herein, the term “package” refers to asuitable solid matrix or material such as glass, plastic, paper, foil,and the like, capable of holding the individual kit components. Thus,for example, a package can be a plastic container used to containsuitable quantities of an inventive device. The packaging materialgenerally has an external label which indicates the contents and/orpurpose of the kit and/or its components.

Diagnostic Protocols

As disclosed herein, various embodiments include a method of diagnosinga disease and/or condition in a subject by one or more of combinationsof the following steps: (1) obtaining a sample from a subject 200; (2)staining of the sample 201; (3) transfer to a microfluidic device 202,where (4) the carrier solution may pass through while stained sample areretained 203, and then (5) diagnosing the subject by measuring theoptical density and comparing the density to a standard 204.

In one embodiment, the staining of the sample 201 includes drawing bloodfrom a subject into a centrifuge tube containing anti-coagulant. Inanother embodiment, the blood is centrifuged, followed by aspirating thePRP (platelet-rich plasma) layer, acidifying the PRP by initially addingACD, then adding the ACD dropwise while checking the pH of the PRP. Inanother embodiment the PRP is diluted with Phosphate Buffered Saline(PBS), centrifuged, and then supernatant transferred. In anotherembodiment, the supernatant is centrifuged to pellet the platelets andthen resuspended in Modified Tyrode's Buffer. In another embodiment, theplatelets in Modified Tyrode's Buffer are added to tubes with anequivalent volume of 2× reaction staining solution. In anotherembodiment, the tubes are incubated, centrifuged, resulting pelletsresuspended, and then transferred to a microfluidic and/or centrifugedevice for quantification.

In one embodiment, the transfer of stained samples to the injector port102 of the microfluidic device for quantification may be by manualinjection such as via syringe. In another embodiment, the transfer maybe in an automated fashion, as by infusion pump or other device. Inanother embodiment, the stained platelet suspension will flow, underpressure of application (e.g., syringe or pump) through an internalchannel(s) to the optical measurement chamber 104, which overlies thefiltration channels 107. As described herein, the filtration channels107 may be aligned to maximize platelet carrier solution (i.e., buffer)pass-through while retaining the platelets in the optical measurementchamber 104. In another embodiment, the filtration channels 107 aredesigned to enhance carrier solution pass-through via capillary forcesand thereby enhance platelet packing density in the optical measurementchamber 104. Thus, in another embodiment, the carrier solution drainageand concomitant platelet packing may be achieved via two forces: theforce of application and capillary force in the drainage channels. Asthe carrier solution is pulled from the optical measurement chamber 104,platelet packing density reaches a maximal, and thus constant, value,suitable for the control of subsequent measurements of staining densitywithin the platelets.

In another embodiment, the optical density of the optical measurementchamber 104 may be measured at an established wavelength and the resultcompared against a tabled value. In another embodiment, the opticaldensity of the optical measurement chamber 104 may be measured inconcert with an included standard reference material. This measurementcan be achieved, for example, by housing the microfluidics apparatus, ormultiple apparatus for greater economy, in a manufactured plate or framewith the dimensions of a standard microplate (e.g., 96-well microplate),with the optical measurement chamber 104 aligned with standardcoordinates of a multiwell microplate. Thus, in another embodiment, onemay use a standard microplate reader to select the specified wellcoordinates and specified wavelength as they are accustomed, and performmeasurement. Another embodiment may incorporate the microfluidicsapparatus on the vertical and housed in a frame with the dimensions of astandard spectrophotometer cuvette, such that the optical measurementchamber 104 aligned with the standard beam of a spectrophotometer. Indesigns targeting measurement in a spectrophotometer orspectrophotometric microplate reader, spectrophotometric measurement atdesignated wavelengths is used as a proxy for densitometric measurement.Thus, in another embodiment, alternate wavelengths could be designated,and may or may not incorporate various correction factors, for use innon-monochromator-equipped spectrophotometric devices or when thedesignated wavelength cannot be otherwise used. Another embodiment mayincorporate similar microfluidics design characteristics but enablingdirect measurement in an optical densitometer. Each described embodimentwould be equally valid and each could be provided as a kit targeted tothe user's available device (e.g., “diagnostic kit for microplatereaders”, “diagnostic kit for spectrophotometers”).

As further disclosed herein, various embodiments include a method ofdiagnosing a disease and/or condition in a subject by one or more ofcombinations of the following steps: (1) a sample may be obtained fromthe subject 200; (2) followed by staining of the sample 201; and (3)transfer of the stained sample to a centrifugal device 205; (4)centrifuged to induce sedimentation of stained sample 206; and then (5)measuring the optical density and comparing the density to a standard207.

In another embodiment, the transfer of the stained sample to acentrifugal device may include transfer to a tube insert as described inFIG. 3 herein. In another embodiment, the tube insert is placed within astandard 1.5 ml microcentrifuge tube 110, the stained plateletsuspension is transferred (e.g., via pipette) into the tube insert, andthe tube and insert are centrifuged at a determined speed (e.g., 2000×g)for a determined time (e.g., 15 min) to induce the sedimentation of thestained platelets into the optical measurement chamber 104, or “opticalpocket”. Thus, under constant centrifugal force, stained plateletpacking density within the measurement chamber reaches a maximal, andthus constant, value, suitable for the control of subsequentmeasurements of staining density within the platelets.

In another embodiment, the optical density of the optical measurementchamber is measured at an established wavelength (e.g., 330 nm) and theresult compared against a tabled value, or in concert with an includedstandard reference material. In another embodiment, this measurement canbe achieved, for example, by providing a manufactured plate or framewith the dimensions of a standard microplate (e.g., 96-well microplate),with the measurement chamber aligned with standard coordinates of amultiwell microplate by way of providing for the insertion of the tubeinsert into the frame. Thus, in any standard microplate reader, the usermay select the specified well coordinates and specified wavelength asthey are accustomed, and perform measurement. Another embodiment mayincorporate a frame with the dimensions of a standard spectrophotometercuvette 111, such that the optical measurement chamber 104 is alignedwith the standard beam of a spectrophotometer upon insertion. In designstargeting measurement in a spectrophotometer or spectrophotometricmicroplate reader, spectrophotometric measurement at designatedwavelengths is used as a proxy for densitometric measurement. Thus, inanother embodiment, alternate wavelengths could be designated, and mayor may not incorporate various correction factors, for use innon-monochromator-equipped spectrophotometric devices or when thedesignated wavelength cannot be otherwise used. Another embodiment mayincorporate similar microfluidics design characteristics but enablingdirect measurement in an optical densitometer. Each described embodimentwould be equally valid and each could be provided as a kit targeted tothe user's available device (e.g., “diagnostic kit for microplatereaders”, “diagnostic kit for spectrophotometers”).

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Kit Design to Provide Simple and Low Cost HistochemicalAssessment

Recent research has demonstrated that certain neurological declines(such as in Alzheimer's Disease), can be detected in peripheral cells,such as platelets. By histochemically staining the activity of an enzymeof interest without activating the platelets (i.e., keep whole plateletsin suspension throughout the procedure), the platelets can be forcedinto a tissue-like orientation for densitometric measurement of reactionproduct using a microfluidic device, without relying on the complicatedmethodology needed for activation and adherence. This in turn removesthe need for a secondary control assay, and there is a reduction in thenumber of separate reagents and other supplies necessary, furtherreducing the overall cost and complication of the assay. For instance,the inventors can achieve a pure platelet preparation using differentialcentrifugation in generic low-cost tubes (e.g., evacuated ACD tubes;tested and confirmed by staining for contamination), rather thanrequiring a commercial separation tube with separation media (e.g.,Sigma-Aldrich Accuspin, Vacutainer CPT). The inventors have tested andeliminated common anti-platelet activation factors prostaglandin-1 andapyrase, relying only on acid-citrate-dextrose anticoagulant.

The inventors have designed a microfluidics device that captures/filtersstained platelets, in a uniform array within an optical measurementchamber, while allowing flow-through of the carrier buffer, measuringthe staining product with highest sensitivity. The device providesunprecedented sensitivity in the measurement of the staining product,and thus, unprecedented sensitivity and accuracy in the measurement ofthe enzyme activity itself. This device can also be used as ahigh-quality cell filter, with or without simple modifications, foroptical measurement of any number of modalities (e.g., fluorescence,luminescence) across many types of suspension cells (e.g., lymphocytes,cancer cells). Thus, the number of potential applications for a deviceof this type, both in the clinic and in the laboratory, is nearlyendless.

Example 2 Microfluidics Plate Design

The general microfluidics plate design allows densitometric measurementof the stained suspended-cell sample. The general design incorporatesmicrofabrication technology, including a sandwich design where surfaceetched features become internal features when assembled. The device mayhave, for example, two etched glass plates that, when assembled andbonded, form an internal network of interconnected channels andchambers. The upper plate may contain an inlet port for injection of thesample suspended in buffer, as well as a flow-through channel leading toa measurement chamber. The measurement chamber, or optical measurementchamber, lies over microfiltration channels etched into the lower plate.Six to eight of these devices may, for example, be arrayed on a chipdesigned to the format of 96-well plates, to be read in a standardmicroplate reader.

Example 3 Design Characteristics of the Microfluidics Plate Design

With the design primarily a simple flow-through application, thecritical test parameters are flow and filtration, i.e., the lack of‘clogging’ and the ability to create a uniform measurement field. Intesting of the platelet histochemistry, maintenance of anti-coagulantsuccessfully prevented the aggregation and activation of plateletsthroughout the histochemical procedure. An additional parameter is thefiltration of carrier buffer from the measurement chamber, such that theplatelets form a homogenous layer amenable to densitometric measurement.This is key to one of the simplest variant of this design: to obtain auniform array of platelets within the measurement chamber, the user mayforgo the use of a secondary control assay, relying only on “plateletdensity” as an internal control.

a. The inlet port may be of the type commonly used in microfabricationdesign.

b. The flow channel. The flow channel will allow the unhindered flow ofsuspension to the measurement chamber. Modifications to the flow channelcould be utilized, for example, such as the incorporation ofmicrofabricated pressure control valves that may prove useful inestablishing the consistency of platelet packing density.

c. The measurement chamber. In one embodiment, the dimensions of themeasurement chamber require the device's fabrication from a material ofhigh stiffness (e.g., glass rather than commonly microfabricatedplastics) for two main reasons: 1) reliable platelet packing densitycannot be achieved in a flexible chamber, 2) the proposed dimensions(diameter:height/depth) could lead to collapse of the chamber withsofter materials. In one embodiment, the diameter of the chamber couldbe a maximum of 3 mm. Measurement of the reaction product in the visualspectrum requires optical clarity above and below the sample, thus glassmay be used. An important principal in absorption or densitometricassessment is the scatter of light from an irregular surface (i.e., thetissue); in tissue staining applications described herein, this isminimized by the use of glass slides and coverslips, and the macro-scaleassessments typically performed (e.g., brain regional analysis) arerelatively insensitive to any residual scatter. With a desire to measurethe optical density of the staining product in standard, yet moresensitive, lab equipment such as a microplate reader, rather than anovel assembly of light source, optics, and detector that would have tobe purchased by the enduser, light scatter by an irregular samplesurface becomes much more significant. In one embodiment, this may beaddressed in one of two ways: 1) minimizing the thickness of the tissuethrough which the beam must pass (the depth of the chamber), and 2)utilizing the glass surfaces of our device to force the sample intonear-perfect perpendicular and regular alignment to the beam.

d. Microfiltration. Microfiltration channel devices are designed topassively filter plasma from whole blood, for downstream chemistry,using only capillary action. The design allows reliable filter ofplatelets in suspension, capturing them in the measurement chamber,uniformly without clogging, at any given unit of application. Also,rather than rely only on the front-end application pressure (injectionforce) to drive cell packing, one could also leverage capillary forceson the back-end, to “pull” the platelet suspension into the chamber andmaximize packing uniformity. Thus, rather than simply existing as asimple slotted drain or filter, the filtration channels on the lowerplate can take on an active role in preventing clogging and increasingcell density uniformity.

Example 4 Studies Demonstrating Applicability of Technique

First, assessments were made of maintenance of platelets in suspension.The degree of anti-coagulation necessary to maintain platelets withoutactivation and binding has been assessed, and the redundant applicationof platelet inhibitory factors such as apyrase and prostaglandin (PGE1)has been dropped. This further reduces both the complexity and the costof this assay. Secondly, non-cytochrome c oxidase-mediated DABreactivity was assessed. Peroxidase activity is the largest contributorto background DAB reactivity in most DAB labeling applications, but theinventors' testing utilizing potassium cyanide inhibition of cytochromec oxidase to reveal other reaction mechanisms has indicated that itsactivity is relatively low (and is lowered by increasing plateletwashes).

Example 5 Measurement of Cytochrome C Oxidase Activity—UnprocessedSample Staining

10 mL whole is blood drawn from a test subject into a centrifuge tube ortubes, containing anti-coagulant. The blood is centrifuged at 300 RCFfor 15′, then the PRP (platelet-rich plasma) layer is aspirated to 15 mLtube(s).

Preparation of ACD Anticoagulant Step No. Action 1 Dissolve 1.32 g ofsodium citrate in 85 ml of distilled water. 2 Dissolve 0.48 g of citricacid in the solution from step 1. 3 Dissolve 1.47 g of dextrose in thesolution from step 2. 4 Add distilled water to 100 ml. 5 Filtersterilize through 0.2 um filter.

The PRP is acidified to pH 6.5 by initially adding 300 μL of ACD, thenadding the ACD dropwise while checking the pH of the PRP. Next, the PRPis diluted to 5 mL with Phosphate Buffered Saline (PBS). The PRP is thencentrifuged at 600 RCF for 10′ at room temperature. The supernatant isthen transferred to a new 15 mL tube, and the pellet is discarded.

The supernatant is centrifuged at 2000 RCF for 10′ to pellet theplatelets, which are then resuspended in 300 μL/8 ml blood draw ofModified Tyrode's Buffer prepared as follows:

Preparation of Modified Tyrode's Buffer Step No. Action 1 Dissolve 0.8 gof NaCl in 85 ml of distilled water. 2 Dissolve 0.02 g of KCl in thesolution from step 1. 3 Dissolve 0.02 g of CaCl₂ in the solution fromstep 2. 4 Dissolve 0.01 g of MgCl₂•6H₂O in the solution from step 3. 5Dissolve 0.005 g of NaH₂ PO₄ in the solution from step 4. 6 Dissolve 0.1g of NaHCO₃ in the solution from step 5. 7 Dissolve 0.1 g of glucose inthe solution from step 6. 8 Add distilled water to 100 ml. 9 Filtersterilize through 0.2 um filter.

The staining solutions (at 2X) are prepared as follows:

Preparation of 2X Staining solutions Step No. Action 1 Measure 8 mL of20 mM PBS. 2 Dissolve 0.0012 g of cytochrome c in the liquid fromstep 1. 3 Add 40 μL dimethyl sulfoxide (DMSO) to the solution from step2; mix well. This solution will be used for the control reactions. 4 To4 mL of the solution from step 3 is added 0.004 g diaminobenzidine(DAB); mix well.The reaction staining solution is then heated to 37 C.

Platelets in Tyrode's are added to tubes with an equivalent volume of 2×reaction staining solution. The tubes are closed and incubated for 1hour in a 37 C water bath. The tubes are then centrifuged at 1600 RCFfor 10′ at room temperature.

The resulting pellets are resuspended, and may then be transferred toeither microfluidic or centrifugal device for quantification.

Example 6 Measurement of Cytochrome C Oxidase Activity—Transfer toMicrofluidic Device

After following the protocol as outlined in Example 5 above, theresulting pellets are resuspended and transferred to the microfluidicdevice described herein. The application of stained platelets can be,for example, by manual injection via syringe, or in an automatedfashion, as by infusion pump or other device. The stained plateletsuspension will flow, under pressure of application (e.g., syringe orpump) through the internal channel(s) to the measurement chamber, whichoverlies the microfiltration channels. The microfiltration channels arealigned to maximize platelet carrier solution (i.e., buffer)pass-through while retaining the platelets in the measurement chamber.The microfiltration channels are designed to enhance carrier solutionpass-through via capillary forces and thereby enhance platelet packingdensity in the measurement chamber. Thus, the carrier solution drainageand concomitant platelet packing is achieved via two forces: the forceof application and capillary force in the drainage channels. Thus, asthe carrier solution is pulled from the measurement chamber, plateletpacking density reaches a maximal, and thus constant, value, suitablefor the control of subsequent measurements of staining density withinthe platelets.

Next, the optical density of the optical measurement chamber is measuredat an established wavelength (e.g., 330 nm) and the result comparedagainst a tabled value, or in concert with an included standardreference material. This measurement can be achieved, for example, byhousing the microfluidics apparatus, or multiple apparatus for greatereconomy, in a manufactured plate or frame with the dimensions of astandard microplate (e.g., 96-well microplate), with the measurementchamber aligned with standard coordinates of a multiwell microplate.Thus, in any standard microplate reader, the user may select thespecified well coordinates and specified wavelength as they areaccustomed, and perform measurement. Another embodiment may incorporatethe microfluidics apparatus on the vertical and housed in a frame withthe dimensions of a standard spectrophotometer cuvette, such that themeasurement chamber aligned with the standard beam of aspectrophotometer. In designs targeting measurement in aspectrophotometer or spectrophotometric microplate reader,spectrophotometric measurement at designated wavelengths is used as aproxy for densitometric measurement. Alternate wavelengths could bedesignated, and may or may not incorporate various correction factors,for use in non-monochromator-equipped spectrophotometric devices or whenthe designated wavelength cannot be otherwise used. Another embodimentmay incorporate similar microfluidics design characteristics butenabling direct measurement in an optical densitometer. Each describedembodiment would be equally valid and each could be provided as a kittargeted to the user's available device (e.g., “diagnostic kit formicroplate readers”, “diagnostic kit for spectrophotometers”).

Example 7 Measurement of Cytochrome C Oxidase Activity—Transfer toCentrifugal Device

After following the protocol as outlined in Example 5 above, theresuspended pellets are transferred to the microcentrifuge tube insertdescribed herein. The tube insert is placed within a standard 1.5 mlmicrocentrifuge tube. The stained platelet suspension is transferred(e.g., via pipette) into the insert and the tube is capped. The tube andinsert are then centrifuged at the determined speed (e.g., 2000×g) forthe determined time (e.g., 15 min) to induce the sedimentation of thestained platelets into the measurement chamber, or “optical pocket”.Thus, under constant centrifugal force, stained platelet packing densitywithin the measurement chamber reaches a maximal, and thus constant,value, suitable for the control of subsequent measurements of stainingdensity within the platelets.

Next, the optical density of the optical measurement chamber is measuredat an established wavelength (e.g., 330 nm) and the result comparedagainst a tabled value, or in concert with an included standardreference material. This measurement can be achieved, for example, byproviding a manufactured plate or frame with the dimensions of astandard microplate (e.g., 96-well microplate), with the measurementchamber aligned with standard coordinates of a multiwell microplate byway of providing for the insertion of the tube insert into the frame.Thus, in any standard microplate reader, the user may select thespecified well coordinates and specified wavelength as they areaccustomed, and perform measurement. Another embodiment may incorporatea frame with the dimensions of a standard spectrophotometer cuvette (asshown in FIG. 4 herein), such that the measurement chamber aligned withthe standard beam of a spectrophotometer upon insertion. In designstargeting measurement in a spectrophotometer or spectrophotometricmicroplate reader, spectrophotometric measurement at designatedwavelengths is used as a proxy for densitometric measurement. Alternatewavelengths could be designated, and may or may not incorporate variouscorrection factors, for use in non-monochromator-equippedspectrophotometric devices or when the designated wavelength cannot beotherwise used. Another embodiment may incorporate similar microfluidicsdesign characteristics but enabling direct measurement in an opticaldensitometer. Each described embodiment would be equally valid and eachcould be provided as a kit targeted to the user's available device(e.g., “diagnostic kit for microplate readers”, “diagnostic kit forspectrophotometers”).

Example 8 Assay Optimization

Optimization of assays described herein may include 1) increasingsignal-to-noise (reducing background, increasing sensitivity ofmeasurement), 2) reducing costs (discarding redundant or unnecessarychemical additions), and 3) reducing time (speeding the reaction rate).

a. Reaction Linearity Toward Optimizing Staining Substrate and Time.

One critical difference between the application of DAB staining forlocalization of an enzyme and DAB staining for quantification of enzymeactivity is the intensity to which the stain is driven. For any reliablequantification, such as in routine tissue histochemical assessments, thedegree of DAB staining is held to a nonsaturating level. In contrast,many investigators maximize DAB staining to merely mark the presence ofenzyme, akin to immunological techniques. Use of DAB as described hereinenables its use as a continuous and quantitative, not merely aqualitative, marker; the densitometric results obtained could belinearly converted to enzymatic turnover (i.e., for example, moles ofcytochrome c enzymatically reduced). Based on this premise, the assaymay be tested for linearity of reaction with time, after any significantmodifications to the staining parameters. One may seek to maximizesignal

without saturating; any “plateauing” or reduction of slope of theoptical density over time will indicate saturation, and staining timewill be scaled back. Cobalt chloride or other metals for intensificationmay also be tested as a DAB intensifier and incorporated into theprotocol if it shortens staining time while maintaining/increasingsignal and linearity of optical density with time.

b. Cell Permeabilization.

Histochemical staining with DAB is intensified through the use of DMSO,allowing increased cell permeability while maintaining membraneintegrity. One may assess whether DMSO is required and if increasingDMSO during staining allows for shorter incubation times. Linearity ofthe reaction can also be assessed as above after such changes.

c. Enzyme Specificity.

This may be tested through specific Cytochrome c Oxidase inhibition withpotassium cyanide (KCN). KCN is added simultaneous to the DAB incomparison assays and any residual DAB reactivity is consideredbackground, nonspecific activity.

Example 9 Alternatives and Modifications to Design

a. Internal Control Assay for Cell Number or Content.

If uniform cell packing density cannot be reliably achieved, one mayalso use, in conjunction with various embodiments of the device, asecondary assay to be applied simultaneously, for measurement ofmitochondrial content or similar variables. The choices are numerous.Histochemical staining for succinate dehydrogenase is a logical choice,as the combination of succinate dehydrogenase/cytochrome c oxidasestaining is often used in the determination of mitochondrial disease inmuscle fibers. Succinase dehydrogenase is typically stained withnitroblue tetrazolium, which yields a blue stain, which may be able tospectrophotometrically distinguish from the brown diaminobenzidinereaction generated from cytochrome c oxidase. However, another choicewould be another modality, such as fluorescence or luminescence, thatwould not interfere with the overall optical density of the preparation.Mitotracker Green FM (Invitrogen), for instance, may be employedreliably in this application. It can be quickly applied to cells insuspension, where it has been found that it stains platelets generally(likely due to the dense platelet internal membrane system), and thuscould potentially provide a cell density measure. In other cell types,it is more likely to fluoresce only in mitochondria, and thus provide areasonable mitochondrial density measure. These are but two examplesconsidered, but if cell density is insufficient as a control, one couldexpect to be able to substitute another control in due course withoutany extensive delay.

b. Internal Control Assay for Staining Intensity.

If one is able to rely on a single staining modality (e.g., DAB), onestill must consider the variability in staining outcome due, in mostcircumstances, to operator error. Incorrect incubation temperature islikely the most common error that would not necessarily lead to absolutefailure of the assay and thus quick detection of incorrect results.While the extremes of the range of possible optical density values canbe easily ruled out as errors (i.e., very light stain=low temperature;very dark stain=high temperature), milder errors could result in falsenegatives or false positives. Therefore, one might assess the truetemperature sensitivity of the assay to allow for correction (to whichincubator/water bath temperature logs could be applied). Additionally,one could include a temperature-sensitive artificial reactant (e.g.,peroxidase contained within a permeable matrix) that could be stainedside-by-side with the patient samples, either as an indicator of properconditions, or as an internal correction factor for the assay results.This would, however, increase the complexity and cost of the assay.

Example 10 Histochemical Measurement of Cytochrome C Oxidase ActivityUsing 96-Well Plates

10 mL whole blood is drawn from a test subject into a centrifuge tube.The blood is centrifuged at 300 RCF for 15′, then the PRP (platelet-richplasma) layer is aspirated to 15 mL tube(s).

Preparation of ACD Anticoagulant Step No. Action 1 Dissolve 1.32 g ofsodium citrate in 85 ml of distilled water. 2 Dissolve 0.48 g of citricacid in the solution from step 1. 3 Dissolve 1.47 g of dextrose in thesolution from step 2. 4 Add distilled water to 100 ml. 5 Filtersterilize through 0.2 um filter.

The PRP is acidified to pH 6.5 by initially adding 300 μL ACD, thenadding the ACD dropwise while checking the pH of the PRP. Next, the PRPis diluted to 5 mL with Phosphate Buffered Saline (PBS). The PRP is thencentrifuged at 500 RCF for 10′ at room temperature. The supernatant isthen transferred to a new 15 mL tube, and the pellet is discarded.

The supernatant is centrifuged at 2000 RCF for 10′ to pellet theplatelets, which are then resuspended in 300 μL/8 ml blood draw ofModified Tyrode's Buffer prepared as follows:

Preparation of Tyrode's buffer Step No. Action 1 Dissolve 0.8 g ofsodium chloride in 85 ml of distilled water. 2 Dissolve 0.02 g ofpotassium chloride in the solution from step 1. 3 Dissolve 0.02 g ofcalcium chloride in the solution from step 2. 4 Dissolve 0.01 g ofMgCl₂•6H₂O in the solution from step 3. 5 Dissolve 0.005 g of NaH₂ PO₄in the solution from step 4. 6 Dissolve 0.1 g of NaHCO₃ in the solutionfrom step 5. 7 Dissolve 0.1 g of glucose in the solution from step 6. 8Add distilled water to 100 ml. 9 Filter sterilize through 0.2 um filter.

The staining solutions (at 2×) are prepared as follows:

Preparation of 2X Staining solutions Step No. Action 1 Measure 8 mL of20 mM PBS. 2 Dissolve 0.0012 g of cytochrome c in the liquid fromstep 1. 3 Add 40 μL dimethyl sulfoxide (DMSO) to the solution from step2; mix well. This solution will be used for the control reactions. 4 To4 mL of the solution from step 3 is added 0.004 g diaminobenzidine(DAB); mix well.

The reaction staining solutions are then heated to 37 C. Platelets inTyrode's are added to tubes containing an equivalent volume of 2×reaction staining solution. The tubes are closed and incubated for 1hour in a 37 C water bath. The tubes are then centrifuged at 1600 RCFfor 10′ at room temperature.

The resulting pellets are resuspended and 40 μl is transferred to theround-bottom microplate wells in a 96-well plate. The microplates arerefrigerated and the individual wells are allowed to sediment overnight.

Next, the OD is measured at the determined wavelength (e.g., 330 nm) andthe result compared against a tabled value.

Example 11 Histochemical Measurement of Cytochrome C Oxidase ActivityUsing Microfilter Devices

8.5 mL whole blood is drawn from a test subject into a centrifuge tube.The tube is centrifuged at 300 RCF for 15′, then the PRP (platelet-richplasma) layer is aspirated to a 15 mL tube.

Preparation of ACD Anticoagulant Step No. Action 1 Dissolve 1.32 g ofsodium citrate in 85 ml of distilled water. 2 Dissolve 0.48 g of citricacid in the solution from step 1. 3 Dissolve 1.47 g of dextrose in thesolution from step 2. 4 Add distilled water to 100 ml. 5 Filtersterilize through 0.2 um filter.

The PRP is acidified to pH 6.5 by initially adding 300 μL of ACD, thenadding the ACD dropwise while checking the pH of the PRP. Next, the PRPis diluted to 5 mL with Phosphate Buffered Saline (PBS). The PRP is thencentrifuged at 600 RCF for 10′ at room temperature. The supernatant isthen transferred to a new 15 mL tube, and the pellet is discarded.

The supernatant is centrifuged at 2000 RCF for 10′ to pellet theplatelets, which are then resuspended in 300 μL/8 ml blood draw ofModified Tyrode's Buffer prepared as follows:

Preparation of Modified Tyrode's Buffer Step No. Action 1 Dissolve 0.8 gof NaCl in 85 ml of distilled water. 2 Dissolve 0.02 g of KCl in thesolution from step 1. 3 Dissolve 0.02 g of CaCl₂ in the solution fromstep 2. 4 Dissolve 0.01 g of MgCl₂•6H₂O in the solution from step 3. 5Dissolve 0.005 g of NaH₂ PO₄ in the solution from step 4. 6 Dissolve 0.1g of NaHCO₃ in the solution from step 5. 7 Dissolve 0.1 g of glucose inthe solution from step 6. 8 Add distilled water to 100 ml. 9 Filtersterilize through 0.2 um filter.

The staining solutions (at 2×) are prepared as follows:

Preparation of 2X Staining solutions Step No. Action 1 Measure 8 mL of20 mM PBS. 2 Dissolve 0.0012 g of cytochrome c in the liquid fromstep 1. 3 Add 40 μL dimethyl sulfoxide (DMSO) to the solution from step2; mix well. This solution will be used for the control reactions. 4 To4 mL of the solution from step 3 is added 0.004 g diaminobenzidine(DAB); mix well.

The reaction staining solution is then heated to 37 C.

Platelets in Tyrode's are added to tubes containing an equivalent volumeof 2× reaction staining solution. The tubes are closed and incubated for1 hour in a 37 C water bath. The tubes are then centrifuged at 1600 RCFfor 10′ at room temperature.

The resulting pellets are resuspended and 40 μl is transferred to amicrofilter device as described previously. The 40 μl is added to thedevice's fill port.

Next, the OD is measured at the determined wavelength (e.g., 330 nm) andthe result compared against a tabled value.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. Furthermore, it is to be understood that theinvention is solely defined by the appended claims. It will beunderstood by those within the art that, in general, terms used herein,and especially in the appended claims (e.g., bodies of the appendedclaims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations).

Accordingly, the invention is not limited except as by the appendedclaims.

REFERENCES

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1. A method of processing a sample from a subject, the method comprisingthe steps of: (a) obtaining a sample from the subject; (b) adding areagent to the sample; (c) obtaining from the sample a fractioncomprising unlysed cells; (d) combining the platelet-rich fraction witha staining solution; and (e) quantifying an amount or activity of amitochondrial protein within the concentrated fraction from the subject.2. The method of claim 1, wherein the sample is a tissue sample.
 3. Themethod of claim 1, wherein the reagent comprises an anti-coagulant. 4.The method of claim 1, wherein the sample comprises at least one ofwhole blood or bone marrow.
 5. The method of claim 1, wherein thefraction comprises a platelet-rich fraction.
 6. The method of claim 1,wherein obtaining the fraction comprises centrifugation.
 7. The methodof claim 1, wherein the mitochondrial protein comprises a mitochondrialelectron transport chain enzyme.
 8. The method of claim 7, wherein themitochondrial electron transport chain enzyme comprises cytochromeoxidase.
 9. A method of processing a liquid sample from a subjectsuspected of having a neurodegenerative disease, the method comprisingthe steps of: (a) obtaining the liquid sample from the subject suspectedof having a neurodegenerative disease; (b) isolating from the liquidsample a fraction comprising unlysed cells; (c) quantifying the amountor activity of a mitochondrial target moiety in the fraction bytransferring the sample to a centrifugal device to sediment the unlysedcells within the centrifugal device; and (d) comparing the amount oractivity of the mitochondrial target moiety in the fraction with theamount or activity of the mitochondrial target moiety in a standard. 10.The method of claim 9, wherein the mitochondrial target moiety comprisesa mitochondrial electron transport chain enzyme.
 11. The method of claim10, wherein the mitochondrial electron transport chain enzyme comprisescytochrome oxidase.
 12. The method of claim 9 and further comprisingadding a reagent to the liquid sample prior to isolation of thefraction, wherein the reagent comprises an anticoagulant.
 13. The methodof claim 9, wherein the fraction comprises a platelet-rich fraction. 14.The method of claim 9, wherein the liquid sample comprises at least oneof plasma, whole blood, and bone marrow.
 15. The method of claim 9 andfurther comprising staining the fraction prior to step (c).
 16. A methodof processing a liquid sample from a subject suspected of having aneurodegenerative disease, the method comprising the steps of: (a)obtaining a liquid sample from the subject; (b) adding a reagent to theliquid sample; (c) centrifuging the liquid sample to obtain a fractioncomprising unlysed cells; (d) combining the fraction with a stainingsolution, wherein the staining solutions reactions with a targetmitochondrial protein; (e) measuring the amount or activity of thestained target mitochondrial protein in the fraction; and (f) comparingresults of the measurements from the fraction from the subject withmeasurements in a standard.
 17. The method of claim 16 and furthercomprising concentrating the stained fraction within a device comprisingan optical measurement chamber.
 18. The method of claim 17, whereinmeasuring the amount or activity of the stained target mitochondrialprotein occurs within the device.
 19. The method of claim 16, whereinthe reagent comprises an anticoagulant.
 20. The method of claim 16,wherein the target mitochondrial protein comprises a mitochondrialelectron transport chain enzyme.